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Prevalence and Characterization of Shigatoxigenic Escherichia Coli in Broiler Birds in Mymensingh

M. M. Mamun
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. S. Parvej
Osaka City University Graduate School of Human Life Science, Osaka 558-8585, Japan

S. Ahamed
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

J. Hassan
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

K. H. M. N. H. Nazir
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Y. Nishikawa
Osaka City University Graduate School of Human Life Science, Osaka 558-8585, Japan

M. T. Rahman
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Shigatoxigenic Escherichia coli (STEC) are major food-borne pathogens. They transmit to human through contaminated meat and meat products of animals and poultry, and frequently associated with various types of human illness including haemolytic uremic syndrome. This preliminary study showed the prevalence of STEC in 60 cloacal swab samples of live healthy broiler chickens collected randomly when sold at a wholesale market in Mymensingh district of Bangladesh. Isolation and identification of E. coli was carried out using Eosin Methylene Blue (EMB) agar media and 16S rRNA gene specific polymerase chain reaction (PCR). Among the 60 samples, 49 (81.67%) were found positive to E. coli. These E. coli isolates were screened for the detection of STEC by PCR using stx1 and stx2 gene specific primers. Among the 49 positive samples, 5 (10.20%) were found positive for stx1 gene, and 26 (53.06%) were positive for stx2 gene. In addition, 6 (12.24%) isolates were found positive to both stx1 and stx2 genes, and the remaining 12 (22.46%) were negative. The high prevalence of STEC in the broiler chicken alarms the public health impact as the people are always in close contact with these live broiler chickens in the open market as well as processing of meat at home before cooking. However, further studies are required to uncover the major source(s) for the transmission of STEC to human in rural Bangladesh.

Keywords: Shigatoxigenic E. coli, Prevalence, Characterization, Cloacal swab, Broiler

Location: Bacteriology Laboratory at the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh.

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Broiler

Objectives:

This study was undertaken to determine the occurrence of Shigatoxigenic Escherichia Coli (STEC) in cloacal swab samples of healthy broiler chickens sold in rural markets, and evaluation of their public health significance.

Methodology:

Sample collection- The swab samples from cloacae (n=60) were collected randomly from different local live broiler bird markets of Mymensingh district, Bangladesh, following a convenient sampling method. Properly sterile cotton buds were used for the collection of swab samples. After collection, the swab was instantly transferred to nutrient broth and kept in ice box. The swab samples were transported to the Bacteriology Laboratory at the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh. Cultural and biochemical examination- Overnight incubation of the nutrient broth containing swab samples was done at 37°C. Then samples from the nutrient both were cultured on Eosin Methylene Blue (EMB) agar medium. One colony from each EMB plate that showed metallic sheen on EMB agar was subjected to sugar (dextrose, maltose, lactose, sucrose, and mannitol) fermentation, Methyl Red-Voges Proskauer (MR-VP) and indole production tests following the procedures described by Nazir et al. (2005) and Elafify et al. (2016). DNA Extraction and molecular identification- DNA was obtained from the isolates using boiling method. In brief, 100 μl de- ionized water was taken into an Eppendorf of tube. A pure bacterial colony from overnight culture on 37°C of EMB agar was properly mixed with the de-ionized water. The tube was then transferred to boiling water and boiled for 10 minutes then immediately kept on ice for 10 minutes and then centrifuged at 10,000 rpm for 10 minutes. Supernatant was collected and used as DNA template. The DNA sample was kept in -20°C until used. The isolated organisms that were suspected to be E. coli by their cultural and biochemical characteristics were confirmed as E. coli by PCR with primers specific to E. coli 16S rRNA gene. DNA extracted from known E. coli was sued as PCR positive control while water was used as the negative control. PCR was performed following the procedure described by Hassan et al. (2014). A 25 μl reaction contained 1x Taq Polymerase PCR master mix and 0.4 µM final concentration of each primer. Electrophoresis of the PCR products was done using 2% agarose gel. After electrophoresis, the gel was stained for 10 minutes in ethidium bromide for visualization in UV Tran-illuminator. Isolates that were found positive for E. coli 16SrRNA genes were further screened for stx1 and stx2 by PCR with primers specific for E. coli stx1 and stx2 genes with slight modification. There was 25 μl reaction containing 1x Taq Polymerase PCR master mix and 0.4 µM final concentration of the primers (stx1 or stx2). Thermal profile for the detection of stx1 and stx2 consisted of an initial denaturation at 94^oC for 5 min, followed by denaturation at 94^oC for 1 min, annealing for 1 min (61^oC for stx1, 59^oC for stx2) and extension at 72^oC for 1 min (total 30 cycles), with a final extension at 72^oC for 5 min. PCR amplification was performed using a thermo cycler (Eppendorf Personal, Germany). Appropriate PCR positive (DNA from STEC) control and non-template negative control were also used in each PCR reactions.

Source of Information: Bangl. J. Vet. Med. (2016). 14 (1): 5-8

Article Link (Online Journal): DOI: https://doi.org/10.3329/bjvm.v14i1.28809

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The STEC has been successfully detected by PCR from apparently healthy broiler chicken sold in retail market in Mymensingh, Bangladesh. The high prevalence of STEC in the broiler chicken has great public health significance since these birds can be a potential source for human STEC infection.

Knowledge Management: Journal